Nucleic acids encoding signal transduction inhibitors of allergic reactions

ABSTRACT

The present invention provides an isolated polypeptide consisting of amino acids 1-66 of the human tyrosine kinase, Lyn A, in a pharmaceutically acceptable carrier and an isolated polypeptide consisting of amino acids 1-45 of the human tyrosine kinase, Lyn B, in a pharmaceutically acceptable carrier. The present invention also provides isolated nucleic acids encoding the above-described amino acid sequences, as well as vectors comprising the nucleic acids and cells comprising the vectors. The present invention further provides a method of treating or preventing an allergic disorder in a subject, comprising administering any of the above nucleic acids to a cell of the subject under conditions whereby the nucleic acid is expressed in the subject&#39;s cells, thereby treating the allergic disorder. Additionally provided in this invention is a fusion protein comprising either a polypeptide consisting of amino acids 1-66 of the human tyrosine kinase, Lyn A or a polypeptide consisting of amino acids 1-45 of the human tyrosine kinase, Lyn B and a ligand which binds to and is internalized by cells which express a high affinity receptor for IgE on the surface. A method of treating or preventing an allergic disorder in a subject is also provided, comprising administering an effective amount of the above fusion protein to a cell of the subject, whereby the fusion protein treats the subject&#39;s allergic disorder.

This application is a divisional of, and claims the benefit of,application Ser. No. 09/020,116, filed Feb. 6, 1997, now U.S. Pat. No.6,084,063, issued Jul. 4, 2000, which application is hereby incorporatedin its entirety herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to the prevention and treatment of anallergic disorder. In particular, the invention relates to theadministration of polypeptides of the unique domain of the tyrosinekinase, Lyn, to the cells of a subject having, or at risk of having, anallergic disorder. The polypeptides act within the cells to bind thecytoplasmic domain of the high affinity receptor of IgE (FcεRI) andinhibit signaling through the receptor which would result in release ofhistamines and other substances associated with an allergic reaction,thereby preventing or treating an allergic disorder.

2. Background Art

The family of proteins known as the “multichain immune recognitionreceptors” includes the antigen receptors on B and T-lymphocytes and Fcreceptors including the receptor with high affinity for IgE (FcεRI )(1). Highly homologous in structure, all these receptors utilize, atleast in part, a common mechanism to initiate cellular responses:multi-valent interactions with antigen leads to aggregation of thereceptors and is followed by enhanced phosphorylation of tyrosines, inthe Immune-Receptor Tyrosine-based Activation Motifs (ITAMs) within thecytoplasmic domains of the receptor itself, by a receptor-associatedSrc-family kinase (2).

Aggregation of the FcεRI on mast cells initiates a cascade of eventsleading to degranulation and release of mediators responsible for thesymptoms of atopy. Among the earliest events in the FcεRI cascade is thephosphorylation of tyrosines in the ITAMs on the β and γ chains of thereceptor by the Src-family kinase, Lyn.

Several groups have studied the interaction between FcεRI and Lyn kinaseby a variety of techniques and have demonstrated (7-10) a directinteraction between the kinase and the C-terminal cytoplasmic extensionof the receptor's β chain. Previous studies have also demonstrated thattwo forms of Lyn, designated Lyn A and Lyn B, are produced bydifferential mRNA splicing, both of which behave equivalently (5) andbecome equivalently attached to the receptor after chemicalcross-linking (4).

For FcεRI, a “transphosphorylation” mechanism has been demonstrated thataccounts for the earliest cascade events (3,4). Data from these earlierstudies showed that a small percentage of resting (unphosphorylated)receptors are constitutively associated with Lyn and this constitutiveassociation with the kinase is an absolute requirement for the initialphosphorylation of the receptor.

The present invention provides polypeptides comprising the unique regionof the Lyn kinase which bind the C-terminal cytoplasmic domain of the βchain of the FcεRI and inhibit activation of the FcεRI-mediated cascadeof events that induce an allergic response and methods for the use ofthese polypeptides in treating or preventing allergic disorders.

SUMMARY OF THE INVENTION

The present invention provides an isolated polypeptide, in apharmaceutically acceptable carrier, comprising a polypeptide having anamino acid sequence encoded by a nucleic acid which is at least 95%identical to a nucleic acid selected from the group consisting of: a) anucleic acid encoding a polypeptide having the amino acid sequence ofamino acids 1-66 of the human tyrosine kinase, Lyn A; b) a nucleic acidencoding a polypeptide having the amino acid sequence of SEQ ID NO:1; c)a nucleic acid encoding a polypeptide having amino acids 1-10 of thehuman tyrosine kinase, Lyn A; d) a nucleic acid encoding a polypeptidehaving amino acids 1-27 of the human tyrosine kinase, Lyn A; e) anucleic acid encoding a polypeptide having amino acids 27-66 of thehuman tyrosine kinase, Lyn A; and f) a nucleic acid encoding apolypeptide having any five or more contiguous amino acids of aminoacids 1-66 of the human tyrosine kinase, Lyn A, wherein the polypeptidehas substantially the same biologically functional activity of thepolypeptide encoded by the nucleic acid sequence as set forth in (a),(b), (c), (d), (e) or (f).

Also provided is an isolated polypeptide, in a pharmaceuticallyacceptable carrier, comprising a polypeptide having an amino acidsequence encoded by a nucleic acid which is at least 95% identical to anucleic acid selected from the group consisting of: a) a nucleic acidencoding a polypeptide having the amino acid sequence of amino acids1-45 of the human tyrosine kinase, Lyn B; b) a nucleic acid encoding apolypeptide having the amino acid sequence of SEQ ID NO:2; and c) anucleic acid encoding a polypeptide having any five or more amino acidsof amino acids 1-45 of the human tyrosine kinase, Lyn B, wherein thepolypeptide has substantially the same biologically functional activityof the polypeptide encoded by the nucleic acid sequence as set forth in(a), (b) or (c).

Furthermore, the present invention provides an isolated polypeptide,which can be in a pharmaceutically acceptable carrier, produced from acell transformed with a nucleic acid which is at least 95% identical toa nucleic acid selected from the group consisting of: a) a nucleic acidencoding a polypeptide having the amino acid sequence of amino acids1-66 of the human tyrosine kinase, Lyn A; b) a nucleic acid encoding apolypeptide having the amino acid sequence of SEQ ID NO:1; c) a nucleicacid encoding a polypeptide having amino acids 1-10 of the humantyrosine kinase, Lyn A; d) a nucleic acid encoding a polypeptide havingamino acids 1-27 of the human tyrosine kinase, Lyn A; e) a nucleic acidencoding a polypeptide having amino acids 27-66 of the human tyrosinekinase, Lyn A; and f) a nucleic acid encoding a polypeptide having anyfive or more contiguous amino acids of amino acids 1-66 of the humantyrosine kinase, Lyn A, wherein the polypeptide has substantially thesame biologically functional activity of the polypeptide encoded by thenucleic acid sequence as set forth in (a), (b), (c), (d), (e) or (f).

In addition, the present invention provides an isolated polypeptide,which can be in a pharmaceutically acceptable carrier, produced from acell transformed with a nucleic acid which is at least 95% identical toa nucleic acid selected from the group consisting of: a) a nucleic acidencoding a polypeptide having the amino acid sequence of amino acids1-45 of the human tyrosine kinase, Lyn B; b) a nucleic acid encoding apolypeptide having the amino acid sequence of SEQ ID NO:2; and c) anucleic acid encoding a polypeptide having any five or more amino acidsof amino acids 1-45 of the human tyrosine kinase, Lyn B, wherein thepolypeptide has substantially the same biologically functional activityof the polypeptide encoded by the nucleic acid sequence as set forth in(a), (b) or (c).

A method of treating or preventing an allergic disorder in a subject isalso provided, comprising administering the nucleic acid of the presentinvention to a cell of the subject under conditions whereby the nucleicacid is expressed in the subject's cells, thereby treating the allergicdisorder.

The present invention also provides a fusion protein, comprising apolypeptide having an amino acid sequence encoded by a nucleic acidwhich is at least 95% identical to a nucleic acid selected from thegroup consisting of: a) a nucleic acid encoding a polypeptide having theamino acid sequence of amino acids 1-66 of the human tyrosine kinase,Lyn A; b) a nucleic acid encoding a polypeptide having the amino acidsequence of SEQ ID NO:1; c) a nucleic acid encoding a polypeptide havingamino acids 1-10 of the human tyrosine kinase, Lyn A; d) a nucleic acidencoding a polypeptide having amino acids 1-27 of the human tyrosinekinase, Lyn A; e) a nucleic acid encoding a polypeptide having aminoacids 27-66 of the human tyrosine kinase, Lyn A; and f) a nucleic acidencoding a polypeptide having any five or more contiguous amino acids ofamino acids 1-66 of the human tyrosine kinase, Lyn A, wherein thepolypeptide has substantially the same biologically functional activityof the polypeptide encoded by the nucleic acid sequence as set forth in(a), (b), (c), (d), (e) or (f), and a ligand which binds to and isinternalized by cells which express a high affinity receptor for IgE onthe surface.

The present invention additionally provides a fusion protein, comprisinga polypeptide having an amino acid sequence encoded by a nucleic acidwhich is at least 95% identical to a nucleic acid selected from thegroup consisting of: a) a nucleic acid encoding a polypeptide having theamino acid sequence of amino acids 1-45 of the human tyrosine kinase,Lyn B; b) a nucleic acid encoding a polypeptide having the amino acidsequence of SEQ ID NO:2; and c) a nucleic acid encoding a polypeptidehaving any five or more amino acids of amino acids 1-45 of the humantyrosine kinase, Lyn B, wherein the polypeptide has substantially thesame biologically functional activity of the polypeptide encoded by thenucleic acid sequence as set forth in (a), (b) or (c) and a ligand whichbinds to and is internalized by cells which express a high affinityreceptor for IgE on the surface.

Further provided is a fusion protein, produced from a cell transformedwith a nucleic acid which is at least 95% identical to a nucleic acidselected from the group consisting of: a) a nucleic acid encoding apolypeptide having the amino acid sequence of amino acids 1-66 of thehuman tyrosine kinase, Lyn A; b) a nucleic acid encoding a polypeptidehaving the amino acid sequence of SEQ ID NO:1; c) a nucleic acidencoding a polypeptide having amino acids 1-10 of the human tyrosinekinase, Lyn A; d) a nucleic acid encoding a polypeptide having aminoacids 1-27 of the human tyrosine kinase, Lyn A; e) a nucleic acidencoding a polypeptide having amino acids 27-66 of the human tyrosinekinase, Lyn A; and f) a nucleic acid encoding a polypeptide having anyfive or more contiguous amino acids of amino acids 1-66 of the humantyrosine kinase, Lyn A, wherein the polypeptide has substantially thesame biologically functional activity of the polypeptide encoded by thenucleic acid sequence as set forth in (a), (b), (c), (d), (e) or (f),and encoding a ligand which binds to and is internalized by cells whichexpress a high affinity receptor for IgE on the surface.

In addition, the present invention provides a fusion protein, producedfrom a cell transformed with a nucleic acid which is at least 95%identical to a nucleic acid selected from the group consisting of: a) anucleic acid encoding a polypeptide having the amino acid sequence ofamino acids 1-45 of the human tyrosine kinase, Lyn B; b) a nucleic acidencoding a polypeptide having the amino acid sequence of SEQ ID NO:2;and c) a nucleic acid encoding a polypeptide having any five or moreamino acids of amino acids 1-45 of the human tyrosine kinase, Lyn B,wherein the polypeptide has substantially the same biologicallyfunctional activity of the polypeptide encoded by the nucleic acidsequence as set forth in (a), (b) or (c), and encoding a ligand whichbinds to and is internalized by cells which express a high affinityreceptor for IgE on the surface.

Finally, the present invention provides a method of treating orpreventing an allergic disorder in a subject, comprising administeringthe fusion protein of the present invention to a cell of the subject,whereby the fusion protein treats or prevents the subject's allergicdisorder.

Various other objectives and advantages of the present invention willbecome apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D. Receptor and kinase proteins and the constructs used for theyeast two hybrid experiments. 1A: The β chain and the Gal 4 bindingdomain (BD)-receptor subunit fusion proteins based on the cytoplasmicdomains of the subunit. The four transmembrane domains of the subunitare shown shaded. 1B: The γ chain and the Gal 4 binding domain-receptorsubunit fusion proteins based on the cytoplasmic domain of the subunit.The transmembrane domain is shown shaded. 1C: Lyn B and the Gal4-activation domain(ACT)-kinase fusion proteins based on the completekinase or its unique domain. 1D: Lyn A and the Gal 4-activationdomain-kinase fusion proteins based on the complete kinase or its uniquedomain.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “a” can include multiples.

The present invention is based on the surprising discovery that bindingof a polypeptide consisting of only the unique domain of the Lyn kinase,or portions thereof, to the C-terminal cytoplasmic domain of the β chainof the FcεRI results in inhibition of the transduction signalingactivity of the FcεRI. The signaling activity of the FcεRI is triggeredby binding of ligand to the extracellular domain of the receptor andaggregation of ligand-bound receptors on the cell surface of mast cellsand basophils. Upon ligand binding and aggregation, the FcεRI transducesa signal across the cell membrane which initiates a cascade of eventsleading to release of chemical substances such as histamine, serotonin,prostaglandins and cytokines, resulting in the production of an allergicreaction in a subject. By inhibiting the initiation of this cascade ofevents, the release of these substances is blocked. In this manner, anallergic reaction can be treated or prevented at the level ofintracellular inhibition of the production and release ofallergy-inducing substances from the cells, as opposed to conventionaltreatment methods which attempt to block the effects of theallergy-inducing substances after they are released from activatedcells. Thus, by inhibiting an allergic response at an earlier stage, thepresent invention provides a more efficient and effective way to treatand prevent allergic reactions than has been previously achieved.

Thus, the present invention provides an isolated polypeptide consistingof amino acids 1-66 of the tyrosine kinase, Lyn A, which can be thehuman Lyn A kinase and an isolated polypeptide having the amino acidsequence of SEQ ID NO:1 (human Lyn A kinase). Also provided is anisolated polypeptide consisting of amino acids 1-45 of the tyrosinekinase, Lyn B, which can be the human Lyn B kinase and an isolatedpolypeptide having the amino acid sequence of SEQ ID NO:2 (human Lyn Bkinase).

Also provided is an isolated polypeptide, comprising a polypeptidehaving an amino acid sequence encoded by a nucleic acid which is atleast 95% identical to a nucleic acid selected from the group consistingof: a) a nucleic acid encoding a polypeptide having the amino acidsequence of amino acids 1-66 of the human tyrosine kinase, Lyn A; b) anucleic acid encoding a polypeptide having the amino acid sequence ofSEQ ID NO:1; c) a nucleic acid encoding a polypeptide having amino acids1-10 of the human tyrosine kinase, Lyn A; d) a nucleic acid encoding apolypeptide having amino acids 1-27 of the human tyrosine kinase, Lyn A;e) a nucleic acid encoding a polypeptide having amino acids 27-66 of thehuman tyrosine kinase, Lyn A; and f) a nucleic acid encoding apolypeptide having any five or more contiguous amino acids of aminoacids 1-66 of the human tyrosine kinase, Lyn A, wherein the polypeptidehas substantially the same biologically functional activity of thepolypeptide encoded by the nucleic acid sequence as set forth in (a),(b), (c), (d), (e) or (f).

Thus, the polypeptide of this invention can comprise the amino acidsequence of 1) amino acids 1-66 of the human tyrosine kinase, Lyn A, 2)SEQ ID NO:1, 3) amino acids 1-10 of the human tyrosine kinase, Lyn A, 4)amino acids 1-27 of the human tyrosine kinase, Lyn A, 5) amino acids27-66 of the human tyrosine kinase, Lyn A, or 6) any five or morecontiguous amino acids of amino acids 1-66 of the human tyrosine kinase,Lyn A.

In addition, the present invention provides an isolated polypeptide,comprising a polypeptide having an amino acid sequence encoded by anucleic acid which is at least 95% identical to a nucleic acid selectedfrom the group consisting of: a) a nucleic acid encoding a polypeptidehaving the amino acid sequence of amino acids 1-45 of the human tyrosinekinase, Lyn B; b) a nucleic acid encoding a polypeptide having the aminoacid sequence of SEQ ID NO:2; and c) a nucleic acid encoding apolypeptide having any five or more amino acids of amino acids 1-45 ofthe human tyrosine kinase, Lyn B, wherein the polypeptide hassubstantially the same biologically functional activity of thepolypeptide encoded by the nucleic acid sequence as set forth in (a),(b) or (c).

Thus, the polypeptide of this invention can comprise the amino acidsequence of 1) amino acids 1-45 of the human tyrosine kinase, Lyn B, 2)SEQ ID NO:2, or 3) any five or more amino acids of amino acids 1-45 ofthe human tyrosine kinase, Lyn B.

Also provided is an isolated nucleic acid encoding the amino acidsequence of the polypeptides described above, a vector comprising thenucleic acid and cell comprising the vector. Furthermore, thepolypeptides of this invention and the nucleic acids encoding them canbe in a pharmaceutically acceptable carrier.

Furthermore, the present invention provides an isolated polypeptide, ina pharmaceutically acceptable carrier, produced from a cell transformedwith a nucleic acid which is at least 95% identical to a nucleic acidselected from the group consisting of: a) a nucleic acid encoding apolypeptide having the amino acid sequence of amino acids 1-66 of thehuman tyrosine kinase, Lyn A; b) a nucleic acid encoding a polypeptidehaving the amino acid sequence of SEQ ID NO:1; c) a nucleic acidencoding a polypeptide having amino acids 1-10 of the human tyrosinekinase, Lyn A; d) a nucleic acid encoding a polypeptide having aminoacids 1-27 of the human tyrosine kinase, Lyn A; e) a nucleic acidencoding a polypeptide having amino acids 27-66 of the human tyrosinekinase, Lyn A; and f) a nucleic acid encoding a polypeptide having anyfive or more contiguous amino acids of amino acids 1-66 of the humantyrosine kinase, Lyn A, wherein the polypeptide has substantially thesame biologically functional activity of the polypeptide encoded by thenucleic acid sequence as set forth in (a), (b), (c), (d), (e) or (f). Asused herein, “transformed” means a cell into which exogenous nucleicacid has been introduced either as naked DNA or as part of a vector.

The polypeptide of this invention can have from 80 to 100% identity withthe nucleic acid sequences set forth herein and still have substantiallythe same biologically functional activity of the polypeptides encoded bythese nucleic acids. Such variation in nucleic acid sequence amongpolypeptides having the same activity can be due to natural variationwithin a species or among species, as well as a result of modificationof the amino acid sequence, as described below. The biologicallyfunctional activity of a polypeptide having between 80 and 100% identitycan be determined according to the protocols for binding and inhibitingphosphorylation as described herein.

Thus, the present invention also provides a polypeptide produced from acell transformed with a nucleic acid encoding the amino acid sequenceof: 1) amino acids 1-66 of the human tyrosine kinase, Lyn A; 2). SEQ IDNO:1; 3) amino acids 1-10 of the human tyrosine kinase, Lyn A; 4) aminoacids 1-27 of the human tyrosine kinase, Lyn A; 5) amino acids 27-66 ofthe human tyrosine kinase, Lyn A; or 6) any five or more contiguousamino acids of amino acids 1-66 of the human tyrosine kinase, Lyn A.

Also provided is an isolated polypeptide, in a pharmaceuticallyacceptable carrier, produced from a cell transformed with a nucleic acidwhich is at least 95% identical to a nucleic acid selected from thegroup consisting of: a) a nucleic acid encoding a polypeptide having theamino acid sequence of amino acids 1-45 of the human tyrosine kinase,Lyn B; b) a nucleic acid encoding a polypeptide having the amino acidsequence of SEQ ID NO:2; and c) a nucleic acid encoding a polypeptidehaving any five or more amino acids of amino acids 1-45 of the humantyrosine kinase, Lyn B, wherein the polypeptide has substantially thesame biologically functional activity of the polypeptide encoded by thenucleic acid sequence as set forth in (a), (b) or (c).

Thus, the present invention provides a polypeptide produced from a celltransformed with a nucleic acid encoding the amino acid sequence of: 1)amino acids 1-45 of the human tyrosine kinase, Lyn B; 2) SEQ ID NO:2; or3) any five or more contiguous amino acids of amino acids 1-45 of thehuman tyrosine kinase, Lyn B. As used herein, the polypeptide of thisinvention is the unique domain of the tyrosine kinase, Lyn A (aminoacids 1-66) or Lyn B (amino acids 1-45)or any portion thereof.

Thus, the polypeptide of this invention can consist of any portion ofthe amino acid sequence of amino acids 1-66 of Lyn A or of the aminoacid sequence of SEQ ID NO:1, as well as any portion of the amino acidsequence of amino acids 1-45 of Lyn B or of the amino acid sequence ofSEQ ID NO:2. For example, the polypeptide of this invention can be aminoacids 1-10 (SEQ ID NO:3), amino acids 1-27 (SEQ ID NO:4) or amino acids27-66 (SEQ ID NO:5) of Lyn A, all of which have been demonstrated toassociate with the FcεRI βc at levels greater than the negative control(consisting of Lyn A residues 27-66 out of frame (OOF).

It would be well understood by one of skill in the art that any portionof the amino acid sequence of amino acids 1-66 of Lyn A or any portionof the amino acid sequence of amino acids 1-45 of Lyn B can beidentified based on the known sequence of Lyn A and Lyn B (22, 24) andproduced according to methods well known in the art (e.g., peptidesynthesis; expression of synthesized oligonucleotides). The portion ofthe amino acid sequence of Lyn A or of Lyn B can then be identified aseffective in inhibiting the signaling activity of FcεRI according to themethods described in the Examples herein. Thus, the portion of the aminoacid sequence of Lyn A or Lyn B which can be produced and tested forinhibitory activity according the methods of this invention can be anyfive or more contiguous amino acids of amino acids 1-66 of Lyn A or 1-45of Lyn B. For example, the portion of this invention can be amino acids1-5, 15-30, 22-45, etc., of Lyn A or Lyn B.

As used herein, “isolated” and/or “purified” means a polypeptide whichis substantially free from the naturally occurring materials with whichthe polypeptide is normally associated in nature. Also as used herein,“polypeptide” refers to a molecule comprised of amino acids whichcorrespond to those encoded by a nucleic acid. The polypeptides of thisinvention can consist of the entire amino acid sequence of amino acids1-66 of Lyn A or of amino acids 1-45 of Lyn B or portions thereof, asset forth above. The polypeptides or portions thereof of the presentinvention can be obtained by isolation and purification from cells wherethey are produced naturally or by expression of DNA encoding amino acids1-66 of Lyn A or amino acids 1-45 of Lyn B or portions thereof. Thepolypeptides of the present invention or portions thereof can beobtained by chemical synthesis of peptides, by proteolytic cleavage ofthe polypeptides and by synthesis from nucleic acids (either naturallyoccurring or synthesized) encoding the amino acid sequence of interest.The polypeptide may include conservative substitutions where a naturallyoccurring amino acid is replaced by one having similar properties. Suchconservative substitutions do not alter the FcεRI signaltransduction-inhibiting activity of the polypeptide and would beunderstood to include at least those listed in Table 1 (35).

Thus, it is understood that, where desired, modifications and changesmay be made in the nucleic acid and/or amino acid sequence of thepolypeptides of the present invention and still obtain a polypeptidehaving like or otherwise desirable characteristics. Such changes mayoccur in natural isolates or may be synthetically introduced usingsite-specific mutagenesis, the procedures for which, such as mis-matchpolymerase chain reaction (PCR), are well known in the art.

For example, certain amino acids may be substituted for other aminoacids in a Lyn A or Lyn B polypeptide of this invention withoutappreciable loss of FcεRi signal transduction-inhibiting activity. Sinceit is the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in the Lyn A or Lyn B amino acid sequence (or,of course, the underlying nucleic acid sequence) and nevertheless obtaina Lyn A or Lyn B polypeptide of this invention with like properties. Itis thus contemplated that various changes may be made in the amino acidsequence of the Lyn A or Lyn B polypeptide (or underlying nucleic acidsequence) of this invention without appreciable loss of biologicalutility or activity and possibly with an increase in such utility oractivity.

The present invention also provides an isolated nucleic acid encoding apolypeptide consisting of amino acids 1-66 of the human tyrosine kinase,Lyn A and an isolated nucleic acid encoding a polypeptide having theamino acid sequence of SEQ ID NO:1. Also provided is an isolated nucleicacid encoding a polypeptide consisting of amino acids 1-45 of the humantyrosine kinase, Lyn B and an isolated nucleic acid encoding apolypeptide having the amino acid sequence of SEQ ID NO:2. The nucleicacids of this invention can be in a pharmaceutically acceptable carrier.

“Nucleic acid” as used herein refers to single- or double-strandedmolecules which may be DNA, comprised of the nucleotide bases A, T, Cand G, or RNA, comprised of the bases A, U (substitute for T), C and G.The nucleic acid may represent a coding strand or its complement.Nucleic acids may be identical in sequence to the sequence which isnaturally occurring or may include alternative codons which encode thesame amino acid as that which is found in the naturally occurringsequence (34). Furthermore, nucleic acids may include codons whichrepresent conservative substitutions of amino acids as described inTable 1.

As used herein, the term “isolated” means a nucleic acid separated orsubstantially free from at least some of the other components of thenaturally occurring organism, for example, the cell structuralcomponents commonly found associated with nucleic acids in a cellularenvironment and/or other nucleic acids. The isolation of nucleic acidscan therefore be accomplished by techniques such as cell lysis followedby phenol plus chloroform extraction, followed by ethanol precipitationof the nucleic acids (33). The nucleic acids of this invention can beisolated from cells according to methods well known in the art.Alternatively, the nucleic acids of the present invention can besynthesized according to standard protocols well described in theliterature.

The nucleic acid encoding the polypeptide of Lyn A or of Lyn B orportion thereof of this invention can be part of a recombinant nucleicacid comprising any combination of restriction sites and/or functionalelements as are well known in the art which facilitate molecularcloning, expression and other recombinant DNA manipulations. Thus, thepresent invention further provides a recombinant nucleic acid comprisingthe nucleic acid encoding the polypeptide of Lyn A or Lyn B, or portionthereof of the present invention. In particular, the isolated nucleicacid encoding the polypeptide of Lyn A or of Lyn B or portion thereofcan be present in a vector and the vector can be present in a cell,which can be a cell cultured in vitro or a cell in a transgenic animal.

Thus, the present invention further provides a vector comprising anucleic acid encoding the polypeptide of Lyn A or of Lyn B or a portionthereof (e.g., peptides consisting of amino acids 1-10, 1-27 or 27-66 ofLyn A) of this invention. The vector can be in a pharmaceuticallyacceptable carrier. The vector can be an expression vector whichcontains all of the genetic components required for expression of thenucleic acid encoding the polypeptide of Lyn A or of Lyn B or portionthereof in cells into which the vector has been introduced, as are wellknown in the art. The expression vector can be a commercial expressionvector or it can be constructed in the laboratory according to standardmolecular biology protocols. The expression vector can comprise viralnucleic acid including, but not limited to, adenovirus, retrovirus andor adeno-associated virus nucleic acid. The nucleic acid or vector ofthis invention can also be in a liposome or a delivery vehicle which canbe taken up by a cell via receptor-mediated or other type ofendocytosis.

A polypeptide of Lyn A or of Lyn B or portion thereof of the presentinvention which is identified to inhibit the signaling activity of FcεRIaccording to the methods provided herein can be administered to asubject to treat or prevent an allergic disorder. Thus, the presentinvention further provides a method for treating or preventing anallergic disorder in a subject, comprising administering the nucleicacid of this invention, which encodes a polypeptide of Lyn A or of Lyn Bor a portion thereof, to a cell of the subject under conditions wherebythe nucleic acid is expressed in the subject's cells, thereby treatingthe allergic disorder.

The subject can be any animal in which it is desirable to inhibit thesignal transducing activity of an immunoglobulin receptor which mediatesan allergic reaction. In a preferred embodiment, the animal of thepresent invention is a human. In addition, non-human animals which canbe treated by the method of this invention can include, but are notlimited to, cats, dogs, birds, horses, cows, goats, sheep, guinea pigs,hamsters, gerbils and rabbits, as well as any other animal in which thepolypeptide of the present invention can inhibit the signal transducingactivity of an immunoglobulin receptor, thereby treating or preventingan allergic response.

As recited herein, an allergic disorder describes a disease state orsyndrome whereby the body produces a dysfunctional immune response toenvironmental antigens comprising immunoglobulin E (IgE) antibodieswhich evoke allergic symptoms such as itching, sneezing, coughing,respiratory congestion, rhinorrhea, skin eruptions and the like, as wellas severe reactions, such as asthma attacks and systemic anaphylaxis.Examples of allergic diseases and disorders which can be treated orprevented by the methods of this invention include, but are not limitedto, drug hypersensitivity, allergic rhinitis, bronchial asthma, ragweedpollen hayfever, anaphylactic syndrome, urticaria, angioedema, atopicdermatitis, erythema nodosum, erythema multiforme, Stevens-JohnsonSyndrome, cutaneous necrotizing venulitis, bullous skin diseases,allergy to food substances and insect venom-induced allergic reactions(36-39), as well as any other allergic disease or disorder now known oridentified in the future.

As described above, the nucleic acid of the present invention can beadministered in a pharmaceutically acceptable carrier and can bedelivered to the subject's cells in vivo and/or ex vivo by a variety ofmechanisms well known in the art (e.g., uptake of naked DNA, viralinfection, liposome fusion, intramuscular injection of DNA via a genegun, endocytosis and the like).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The nucleic acids of this invention can be introduced intothe cells via any gene transfer mechanism, such as, for example,virus-mediated gene delivery, calcium phosphate mediated gene delivery,electroporation, microinjection or proteoliposomes. The transduced cellscan then be infused (e.g., in a pharmaceutically acceptable carrier) orhomotopically transplanted back into the subject per standard methodsfor the cell or tissue type. Standard methods are known fortransplantation or infusion of various cells into a subject.

The cells of the subject to which the nucleic acid of this invention canbe administered can include any cell which can take up and expressexogenous DNA and which expresses an FcεRI whose C-terminal β chaincytoplasmic domain can be bound by the polypeptide of this invention,the binding of which results in inhibition of the signal transducingactivity of the FcεRI. For example, the cells can be mast cells,basophils and/or eosinophils, as well as any other cell type whichexpresses an FcεRI and in which it would be desirable to inhibit thesignal transducing activity of the FcεRI.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids of the presentinvention can be in the form of naked DNA or the nucleic acids can be ina vector for delivering the nucleic acids to the cells for expression ofthe nucleic acid encoding Lyn A or Lyn B polypeptide or portion thereofinside the cell. The vector can be a commercially available preparation,such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval,Quebec, Canada). Delivery of the nucleic acid or vector to cells can bevia a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the nucleicacid or vector of this invention can be delivered in vivo byelectroporation, the technology for which is available from Genetronics,Inc. (San Diego, Calif.) as well as by means of a SONOPORATION machine(ImaRx Pharmaceutical Corp., Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system which can package a recombinant retroviralgenome (40, 41). The recombinant retrovirus can then be used to infectand thereby deliver to the infected cells nucleic acid encoding thepolypeptide of Lyn A or of Lyn B or a portion thereof. The exact methodof introducing the nucleic acid into mammalian cells is, of course, notlimited to the use of retroviral vectors. Other techniques are widelyavailable for this procedure including the use of adenoviral vectors(42), adeno-associated viral (AAV) vectors (43), lentiviral vectors(44), pseudotyped retroviral vectors (45). Physical transductiontechniques can also be used, such as liposome delivery andreceptor-mediated and other endocytosis mechanisms (see, for example,46). This invention can be used in conjunction with any of these orother commonly used gene transfer methods.

As described above, the nucleic acid or vector of the present inventioncan also be administered in vivo in a pharmaceutically acceptablecarrier. By “pharmaceutically acceptable” is meant a material that isnot biologically or otherwise undesirable, i.e., the material may beadministered to a subject, along with the nucleic acid or vector,without causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier wouldnaturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

The nucleic acid or vector may be administered orally, parenterally(e.g., intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,although topical intranasal administration or administration by inhalantis typically preferred. As used herein, “topical intranasaladministration” means delivery of the nucleic acid or vector into thenose and nasal passages through one or both of the nares and cancomprise delivery by a spraying mechanism or droplet mechanism, orthrough aerosolization of the nucleic acid or vector. The latter may beeffective when a large number of animals is to be treatedsimultaneously. Administration of the nucleic acid or vector by inhalantcan be through the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of the nucleicacid or vector required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every nucleic acid orvector. However, an appropriate amount can be determined by one ofordinary skill in the art using only routine experimentation given theteachings herein (see, e.g., 47).

As one example, if the nucleic acid of this invention is delivered tothe cells of a subject in an adenovirus vector, the dosage foradministration of adenovirus to humans can range from about 10⁷ to 10⁹plaque forming unit (pfu) per injection, but can be as high as 10¹² pfuper injection (48, 49).

Parenteral administration of the nucleic acid or vector of the presentinvention, if used, is generally characterized by injection. Injectablescan be prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution of suspension in liquidprior to injection, or as emulsions. A more recently revised approachfor parenteral administration involves use of a slow release orsustained release system such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795, which is incorporated by referenceherein.

The present invention additionally provides a fusion protein comprisinga polypeptide consisting of amino acids 1-66, or a portion thereof, ofthe human tyrosine kinase, Lyn A or a polypeptide having the amino acidsequence of SEQ ID NO:1, or a portion thereof and a ligand which bindsto and is internalized by cells which express a high affinity receptorfor IgE on the surface.

Also provided is a fusion protein comprising a polypeptide consisting ofamino acids 1-45, or a portion thereof, of the human tyrosine kinase,Lyn B or a polypeptide having the amino acid sequence of SEQ ID NO:2, ora portion thereof and a ligand which binds to and is internalized bycells which express a high affinity receptor for IgE on the surface.

The present invention further provides a fusion protein, comprising apolypeptide having an amino acid sequence encoded by a nucleic acidwhich is at least 95% identical to a nucleic acid selected from thegroup consisting of: a) a nucleic acid encoding a polypeptide having theamino acid sequence of amino acids 1-66 of the human tyrosine kinase,Lyn A; b) a nucleic acid encoding a polypeptide having the amino acidsequence of SEQ ID NO:1; c) a nucleic acid encoding a polypeptide havingamino acids 1-10 of the human tyrosine kinase, Lyn A; d) a nucleic acidencoding a polypeptide having amino acids 1-27 of the human tyrosinekinase, Lyn A; e) a nucleic acid encoding a polypeptide having aminoacids 27-66 of the human tyrosine kinase, Lyn A; and f) a nucleic acidencoding a polypeptide having any five or more contiguous amino acids ofamino acids 1-66 of the human tyrosine kinase, Lyn A, wherein thepolypeptide has substantially the same biologically functional activityof the polypeptide encoded by the nucleic acid sequence as set forth in(a), (b), (c), (d), (e) or (f), and a ligand which binds to and isinternalized by cells which express a high affinity receptor for IgE onthe surface.

The fusion protein of the present invention can comprise a polypeptidecomprising the amino acid sequence of: 1) amino acids 1-66 of the humantyrosine kinase, Lyn A, 2) SEQ ID NO:1, 3) amino acids 1-10 of the humantyrosine kinase, Lyn A, 4) amino acids 1-27 of the human tyrosinekinase, Lyn A, 5) amino acids 27-66 of the human tyrosine kinase, Lyn A,or 6) any five or more contiguous amino acids of amino acids 1-66 of thehuman tyrosine kinase, Lyn A, and a ligand which binds to and isinternalized by cells which express a high affinity receptor for IgE onthe surface.

Further provided is a fusion protein, comprising a polypeptide having anamino acid sequence encoded by a nucleic acid which is at least 95%identical to a nucleic acid selected from the group consisting of: a) anucleic acid encoding a polypeptide having the amino acid sequence ofamino acids 1-45 of the human tyrosine kinase, Lyn B; b) a nucleic acidencoding a polypeptide having the amino acid sequence of SEQ ID NO:2;and c) a nucleic acid encoding a polypeptide having any five or moreamino acids of amino acids 1-45 of the human tyrosine kinase, Lyn B,wherein the polypeptide has substantially the same biologicallyfunctional activity of the polypeptide encoded by the nucleic acidsequence as set forth in (a), (b) or (c) and a ligand which binds to andis internalized by cells which express a high affinity receptor for IgEon the surface.

The fusion protein of the present invention can comprise a polypeptidecomprising the amino acid sequence of: 1) amino acids 1-45 of the humantyrosine kinase, Lyn B, 2) SEQ ID NO:2, or any five or more contiguousamino acids of amino acids 1-45 of the human tyrosine kinase, Lyn B, anda ligand which binds to and is internalized by cells which express ahigh affinity receptor for IgE on the surface.

Additionally, the present invention provides a fusion protein, producedfrom a cell transformed with a nucleic acid which is at least 95%identical to a nucleic acid selected from the group consisting of: a) anucleic acid encoding a polypeptide having the amino acid sequence ofamino acids 1-66 of the human tyrosine kinase, Lyn A; b) a nucleic acidencoding a polypeptide having the amino acid sequence of SEQ ID NO:1; c)a nucleic acid encoding a polypeptide having amino acids 1-10 of thehuman tyrosine kinase, Lyn A; d) a nucleic acid encoding a polypeptidehaving amino acids 1-27 of the human tyrosine kinase, Lyn A; e) anucleic acid encoding a polypeptide having amino acids 27-66 of thehuman tyrosine kinase, Lyn A; and f) a nucleic acid encoding apolypeptide having any five or more contiguous amino acids of aminoacids 1-66 of the human tyrosine kinase, Lyn A, wherein the polypeptidehas substantially the same biologically functional activity of thepolypeptide encoded by the nucleic acid sequence as set forth in (a),(b), (c), (d), (e) or (f), and encoding a ligand which binds to and isinternalized by cells which express a high affinity receptor for IgE onthe surface.

The fusion protein of the present invention can comprise a polypeptideproduced by a cell transformed with a nucleic acid encoding the aminoacid sequence of: 1) amino acids 1-66 of the human tyrosine kinase, LynA, 2) SEQ ID NO:1, 3) amino acids 1-10 of the human tyrosine kinase, LynA, 4) amino acids 1-27 of the human tyrosine kinase, Lyn A, 5) aminoacids 27-66 of the human tyrosine kinase, Lyn A, or 6) any five or morecontiguous amino acids of amino acids 1-66 of the human tyrosine kinase,Lyn A, and a ligand which binds to and is internalized by cells whichexpress a high affinity receptor for IgE on the surface.

In addition, the present invention provides a fusion protein, producedfrom a cell transformed with a nucleic acid which is at least 95%identical to a nucleic acid selected from the group consisting of: a) anucleic acid encoding a polypeptide having the amino acid sequence ofamino acids 1-45 of the human tyrosine kinase, Lyn B; b) a nucleic acidencoding a polypeptide having the amino acid sequence of SEQ ID NO:2;and c) a nucleic acid encoding a polypeptide having any five or moreamino acids of amino acids 1-45 of the human tyrosine kinase, Lyn B,wherein the polypeptide has substantially the same biologicallyfunctional activity of the polypeptide encoded by the nucleic acidsequence as set forth in (a), (b) or (c), and encoding a ligand whichbinds to and is internalized by cells which express a high affinityreceptor for IgE on the surface.

The fusion protein of the present invention can comprise a polypeptideproduced by a cell transformed with a nucleic acid encoding the aminoacid sequence of: 1) amino acids 1-45 of the human tyrosine kinase, LynB, 2) SEQ ID NO:2, or 3) any five or more contiguous amino acids ofamino acids 1-45 of the human tyrosine kinase, Lyn B, and a ligand whichbinds to and is internalized by cells which express a high affinityreceptor for IgE on the surface.

The ligand of the fusion protein can be any ligand which has thecapability of binding to and becoming internalized by cells whichexpress FcεRI or its equivalent in other species, as determined bymethods well known in the art. For example, the ligand of the fusionprotein of this invention can be IgE or portions thereof which can binda receptor, c-Kit or portions thereof which can bind a receptor, or anyother ligand or portion thereof which can bind to and becomeinternalized by cells which express a high affinity receptor for IgE onthe surface.

The present invention further provides a nucleic acid encoding thefusion protein of this invention, a vector comprising the nucleic acidand a cell comprising the vector. The present invention also providesnucleic acids complementary to, or capable of, hybridizing with thenucleic acids encoding the fusion proteins of this invention. The fusionprotein and the nucleic acid encoding it can be in a pharmaceuticallyacceptable carrier.

Protocols for construction of a vector containing a nucleic acidencoding the fusion protein of this invention are well known in the art(see, e.g., 60). For example, nucleic acid encoding a Lyn A or Lyn Bpolypeptide or portion thereof of this invention can be ligated to anucleic acid encoding the ligand of this invention according to standardmolecular biology protocols such that a continuous open reading frameresults which will allow for production of the fusion protein. Thenucleic acids can be obtained from naturally occurring sources or thenucleic acids can be synthesized. The nucleic acid encoding the fusionprotein can be placed into an expression vector, which can be obtainedcommercially or produced in the laboratory.

A variety of vectors and eukaryotic expression systems such as yeast,filamentous fungi, insect cell lines, bird, fish, transgenic plant andmammalian cells, among others, are known to those of ordinary skill inthe art and can be used in the present invention.

Thus, the present invention further contemplates a method of producingthe fusion protein of the present invention, comprising introducing avector encoding the fusion protein into a eukaryotic cell underconditions whereby the nucleic acid encoding the fusion protein isexpressed and the fusion protein is produced; and isolating andpurifying the fusion protein. Isolation and purification of the fusionprotein can be carried out by protocols well known to those of skill inthe art.

The nucleic acid sequences can be expressed in cells after the sequenceshave been operably linked to, i.e., positioned, to ensure thefunctioning of an expression control sequence. These expression vectorsare typically replicable in the cells either as episomes or as anintegral part of the cell's chromosomal DNA. Commonly, expressionvectors can contain selection markers, e.g., tetracycline resistance,hygromycin resistance, gentamicin resistance or methotrexate resistance,to permit detection and/or selection of those cells transformed with thedesired nucleic acid sequences (see, e.g., U.S. Pat. No. 4,704,362).

The cells are rendered competent for transformation by various meansknown in the art. There are several well-known methods of introducingDNA into eukaryotic cells. These include, but are not limited to,calcium phosphate precipitation, fusion of the recipient cells withbacterial protoplasts containing the DNA, treatment of the recipientcells with liposomes containing the DNA, DEAE dextran, electroporation,micro-injection of the DNA directly into the cells, as well as any othertechnique now known or developed in the future for introducing nucleicacid into cells.

The transformed cells are cultured by means well known to one ofordinary skill in the art (54). The expressed polypeptides are isolatedfrom cells grown as suspensions or monolayers. The latter are recoveredby well-known mechanical, chemical, or enzymatic means and purifiedaccording to standard methods well known in the art.

The vector of the invention, e.g., a plasmid, which is used to transformthe host cell, preferably contains nucleic acid sequences to initiatetranscription and sequences to control the translation of the protein.These sequences are referred to as expression control sequences.Suitable vectors for expression systems usually have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase orother glycolytic enzymes, an origin of replication, terminationsequences and the like, as desired. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40 virus,adenovirus, bovine papilloma virus, etc, as are well known in the art.For example, gene sequences to control replication in the host cell maybe incorporated into the vector such as those found in bovine papillomavirus-type vectors (50). A variety of suitable vectors are described inthe literature (51, 52).

Appropriate vectors for expressing proteins in insect cells are usuallyderived from baculovirus. Suitable insect cell lines include, but arenot limited to, mosquito larvae, silkworm, armyworm, moth and Drosophilacell lines such as a Schneider cell line (54), as well as any otherinsect cell line now known or identified in the future to be a suitablehost cell line for baculovirus or other insect cell expression vectors.

When yeast or higher animal host cells are employed, polyadenylation ortranscription terminator sequences from known mammalian genes can beincorporated into the vector. An example of a terminator sequence is thepolyadenylation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (53).

Synthesis of heterologous proteins in yeast is well known. For example,Sherman et al. (55), is a well-recognized work describing the variousmethods available to produce a protein in yeast. Two procedures are usedin transforming yeast cells. In one case, yeast cells are firstconverted into protoplasts using zymolase, lyticase, or glusulase,followed by addition of DNA and polyethylene glycol (PEG). ThePEG-treated protoplasts are then regenerated in a 3% agar medium underselective conditions. Details of this procedure are described by Beggs,J. D. (59) and Hinnen et al. (56). The second procedure does not involveremoval of the cell wall. Instead, the cells are treated with lithiumchloride or acetate and PEG and put on selective plates (57). The fusionproteins of this invention, once expressed, can be isolated from yeastby lysing the cells and applying standard protein isolation andpurification techniques to the lysates. The monitoring of thepurification process can be accomplished by using Western blottechniques or radioinimunoassay or other standard immunoassaytechniques.

Expression of nucleic acid encoding exogenous proteins can also becarried in a variety of mammalian cell lines. Mammalian cells permit theexpression of proteins in an environment that favors importantpost-translational modifications such as folding and cysteine pairing,addition of lipids (including myristate and palmitate), addition ofcomplex carbohydrate structures and secretion of active protein.

Mammalian cell systems can be in the form of monolayers of cells,although mammalian cell suspensions may also be used. A number ofsuitable host cell lines capable of expressing intact proteins have beendeveloped in the art, and include the HEK293, BHK21, COS and CHO celllines, as well as various human cells such as HeLa cells, myeloma celllines, Jurkat cells, etc., as are known in the art. Other animal cellsuseful for the production of proteins are available, for example, fromthe American Type Culture Collection Catalogue of Cell Lines andHybridomas (7th Edition, 1992). Expression vectors for these cells caninclude expression control sequences, such as an origin of replication,a promoter (e.g., the CMV promoter, a HSC tk promoter or pgk[phosphoglycerate kinase] promoter), an enhancer (58) and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites (e.g., an SV40 large T antigen poly Aaddition site) and transcriptional terminator sequences.

The fusion protein of this invention can also be expressed in transgenicplant expression systems known in the art, such as, for example, soybean cells or Nicotiana tabacum cells (63).

It is also contemplated that the nucleic acids encoding the fusionproteins of the present invention can be used to generate transgenicnonhuman animals in which the nucleic acid encoding a fusion protein ofthe present invention is added to the germ line of the animal. Thus, acell of the invention containing an nucleic acid of this invention iscontemplated to include a cell in a transgenic animal. The plantholotoxin or fusion protein can be isolated and purified from materialssecreted by the animal, such as for example, milk secreted from nonhumanmammals. Transgenic animals are generated by standard means known tothose skilled in the art (see, for example, 64).

The fusion protein of the present invention can be in a pharmaceuticallyacceptable carrier, as defined herein and can be administered to asubject according to the same protocols for dosage determination, modesof administration and efficacy determination as set forth aboveregarding the administration of the nucleic acid of this invention to asubject.

Thus, a method of treating or preventing an allergic disorder in asubject is also provided, comprising administering an effective amountof the fusion protein of the present invention to a cell of the subject,whereby the fusion protein treats or prevents the subject's allergicdisorder. In a preferred embodiment, as set forth above, the subject isa human, but can be any animal in which it is desirable to treat orprevent an allergic disorder. Also, the preferred mode of administrationof the fusion protein is intranasally or in an inhalant and the cells towhich the fusion protein is administered are preferably mast cells andbasophils.

It is contemplated that fusion protein of this invention may beadministered orally, parenterally (e.g., intravenously), byintramuscular injection, by intraperitoneal injection, transdermally,extracorporeally, topically or the like, although topical intranasaladministration or administration by inhalant is typically preferred. Asused herein, “topical intranasal administration” means delivery of thefusion protein into the nose and nasal passages through one or both ofthe nares and can comprise delivery by a spraying mechanism or dropletmechanism, or through aerosolization of the fusion protein. The lattermay be effective when a large number of animals is to be treatedsimultaneously. Administration of the fusion protein by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of the fusionprotein required will vary from subject to subject, depending on thespecies, age, weight and general condition of the subject, the severityof the allergic disorder being treated, the particular fusion proteinused, its mode of administration and the like. Thus, it is not possibleto specify an exact amount for every fusion protein. However, anappropriate amount can be determined by one of ordinary skill in the artusing only routine experimentation given the teachings herein (see,e.g., 47).

Parenteral administration of the fusion protein of the presentinvention, if used, is generally characterized by injection. Injectablescan be prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution of suspension in liquidprior to injection, or as emulsions. A more recently revised approachfor parenteral administration involves use of a slow release orsustained release system such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795, which is incorporated by referenceherein.

In the methods of the present invention which describe the treatment ofan allergic disorder with either a nucleic acid or protein, the efficacyof the treatment can be monitored according to clinical protocols wellknown in the art for monitoring the treatment of allergic disorders. Forexample, such clinical parameters as allergy symptoms (itching,sneezing, coughing, respiratory congestion, rhinorrhea, skin eruption,etc.), assays and skin prick tests (wheal and flare response) to knownallergans and serum levels of IgE and allergy-associated cytokines(e.g., interleukin-4, interleukin-5) can be monitored for determiningefficacy. Indicators of efficacy of the treatment can include areduction in severity and/or absence of symptoms, an increase in thenumber of symptom-free days per time period (e.g., per month) and/or areduction in the need for conventional medications such asdecongestants, anti-histamines, mast cell stabilizers andcorticosteroids. Additionally, if the treatment of this invention isdone in conjunction with immunotherapy, efficacy can be evaluated byobserving an increase in tolerated dose of the subject's knownallergan(s). These parameters can be monitored weekly or monthly, aswell as at greater time intervals (e.g., every 3-6 months). In aparticular example, clinical parameters that can be monitored for asthmacan include the number and severity of attacks as determined by symptomsof wheezing, shortness of breath and coughing. The measurement of airwayresistance by the use of respiratory spirometry, the extent ofdisability and the dependence on immunosuppressive medications orbronchodilators can also be determined (61, 62).

Additionally, the efficacy of administration of the nucleic acid orfusion protein of this invention for preventing an allergic disorder ina subject not known to have an allergic disorder, but known to be atrisk of developing an allergic disorder, can be determined by evaluatingclinical parameters such as allergy symptoms (itching, sneezing,coughing, respiratory congestion, rhinorrhea, skin eruption, etc.),assays and skin prick tests (wheal and flare response) to knownallergans and serum levels of IgE and allergy-associated cytokines(e.g., interleukin-4, interleukin-5), over time following administrationof the nucleic acid or fusion protein of this invention. This timeinterval can be very short (i.e, minutes/hours) or very long (i.e.,years/decades). The determination of who would be at risk for thedevelopment of an allergic disorder would be made based on currentknowledge of the known risk factors for a particular allergic disorderas would be familiar to clinicians and researchers in this field, suchas a particularly strong family history of an allergic disorder orexposure to or acquisition of factors or conditions (i.e.,environmental) which are likely to lead to development of an allergicdisorder.

In addition, it would be well understood by the artisan that, on thebasis of the surprising discovery of the present invention, the bindingdomains of other kinases which bind the cytoplasmic domains of receptorsinvolved in signal transduction can be screened for the ability toinhibit signal transduction by the receptor in the same manner by whichthe polypeptide of Lyn A and Lyn B of this invention inhibit the signaltransduction activity of FcεRI. Thus, the present invention alsocontemplates a method of screening polypeptides of kinases for theability to inhibit signal transduction by a receptor having acytoplasmic domain to which the kinase binds comprising producing apolypeptide having the amino acid sequence of a portion of the kinase;determining whether the polypeptide binds the cytoplasmic domain of thereceptor according to the methods herein and determining whether thepolypeptide, which is identified as binding the cytoplasmic domain ofthe receptor, inhibits the signal transducing activity of the receptoraccording to the methods herein, thereby identifying a polypeptide of akinase as having the ability to inhibit signal transduction by thereceptor.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES

Materials. The yeast strains (CG1945 and Y187) and cloning vectors(pAS2-1 and pACT) were obtained from Clontech (Palo Alto, Calif.); theexpression vectors pBlueBac, pCDM8 and pZeo, as well as a baculovirusMAXBAC expression kit were obtained from InVitrogen (Carlsbad, Calif.);polyacrylamide gels used for electrophoresis (PAGE) was obtained fromNOVEX (San Diego, Calif.); the antibiotics (G418, zeocin) were obtainedfrom Life Technologies (Gaithersburg, Md.) and InVitrogen, respectivelyand plasmid DNA purification kits were obtained from Qiagen (SantaClarita, Calif.).

Antibodies. Monoclonal anti-phosphotyrosine antibodies conjugated tohorseradish peroxidase (anti-PY) were obtained from Transduction Labs(PY-20, Lexington, Ky.) or UBI (4G10; Lake Placid, N.Y.). Polyclonalantibodies to human src-family kinases, Lyn and Fyn, were purchased fromUBI and polyclonal antibodies to c-Src and c-Yes were obtained fromSanta Cruz Biotechnology (Santa Cruz, Calif.). Mouse monoclonal anti-DNPIgE (12) and rat IgE (of unknown specificity) (13) were purified asdescribed previously (14, 15) and labeled with carrier-free [¹²⁵I] usingchloramine T (16). Goat anti-mouse IgE was purchased from ICN (CostaMesa, Calif.); rabbit anti-rat IgE was purified as described (17).Covalently cross-linked IgE oligomers were prepared and analyzed asdescribed (6).

Cell Lines. Rat Basophilic Leukemia (RBL-2H3) cells were maintained aspreviously described (18). Chinese Hamster Ovary (CHO) cells were grownin stationary flasks at 37° C. in a humidified atmosphere containing 5%CO₂ in Iscove's Modified EMEM, 10% fetal calf serum, 25 mM HEPES and theappropriate antibiotics to maintain expression of the transfected genes.Spodoptera frugiperda (Sf9) insect cells were maintained in spinnerculture at 27° C. as previously described (19).

DNA Sequencing. The nucleotide sequence of each expression construct wasconfirmed by automated DNA sequencing using a dye terminator kitobtained from Applied Biosystems (Foster City, Calif.).

Isolation of Rat Lyn Kinase cDNAs. A 5′ stretch cDNA library wasprepared from mRNA isolated from RBL cells. Two separate primingreactions with either oligo(dT) or random primers were performed togenerate the first strand. The reactions were pooled prior to secondstrand synthesis. The cDNA library was then prepared in the expressionvector pCDM8 (20). Probes were prepared by restriction digestion ofhuman Lyn A-pSVL. Probes representing the N-terminus (amino acidresidues 1-298) and the C-terminus (residues 163-512) (22) werepurified. The library was plated and colony lifts were hybridized witheither probe. Positive colonies went through secondary and tertiaryscreening. The nucleotide sequence of two clones, designated N14 (2052bp) and C18 (2316 bp), was determined by primer walking and DNAsequencing of both strands. The Wisconsin Package from the GeneticsComputer Group, Inc. was used to assemble and analyze the nucleotidesequences of the isolated clones. N14 contained an open reading frame ofLyn A, beginning with ATG from bp 80 to bp 1616 while clone C18 encodedLyn B beginning with ATG between bp 236 and bp 1709. The sequence of LynA in the coding region was identical to a previously published sequence(21); the sequence of rat Lyn B lacks an “insert” of 21 amino acidsfound in the A form of the kinase at a position identical to thatpreviously shown for human and murine Lyn (22, 23) but is otherwiseidentical to Lyn A. Therefore it differs somewhat from the previouslypublished sequence for rat Lyn B (24).

CHO cells were transiently transfected with the Lyn-pCDM8 plasmids byelectroporation, harvested 48-72 hours later and a lysate of the wholecells was prepared using SDS. After separation by PAGE and transfer,Western blotting with anti-human Lyn confirmed that the expressedproteins had the expected the size for Lyn A (56 kD) and Lyn B (53 kD).

Yeast Two-hybrid Fusion Constructs. To generate DNA binding domainfusion proteins, the N terminal (1-58) and C-terminal (201-243)cytoplasmic domains of the rat FcεRIβ (65) were amplified by polymerasechain reaction (PCR) from the full length cDNA and cloned into theEcoRI/BamHI sites of pAS2-1. The cytoplasmic domain of rat FcεRIγ(residues 27-68), as a PCR fragment, was cloned into Nco I/Bam HI siteof pAS2-1, to generate pAS2-1-γC. To create activation domain fusionproteins, the full length Lyn A and Lyn B and various deletion mutantswere amplified by PCR and cloned into Bam HI/Xho I sites of pACT.

Yeast Two-hybrid Co-transformation, Selective Growth and β-GalactosidaseAssays. Plasmid constructs were introduced into yeast cells by lithiumacetate, following the protocol provided by Clontech. Transformants wereplated on synthetic medium containing 5 mM 3-amino-1,2,4-trizole andlacking leucine, tryptophan and histidine (SD-3) to detect the Hisphenotype, or synthetic medium lacking leucine and tryptophan (SD-2), tomeasure transformation efficiency. The β-galactosidase activity oftransformants was measured in a filter assay with X-gal as substrate orin a liquid assay with an ONPG substrate according to the Clontechprotocol.

Mammalian Transfection Constructs. The 2.3 kb LynB Xho I-digested insertwas isolated from pCDM8 and subcloned into the Xho I site of pZeo. Theunique domain construct was generated by PCR amplification usinginternal sense and antisense primers for the unique domain:5′-CGGGCGGCTCGATGGGATGTATTAAATCAAAAAGGAAAG-3′ (SEQ ID NO:6), and5′-CGGCGGCTCGAGCTAGTCCCCTTGCTCCTCTGGATC-3′ (SEQ ID NO:7), respectively.

The final PCR product was digested with Xho I and cloned back into thepZeo Xho I site. The catalytically inactive Lyn B(K279R)-pZeo constructwas prepared using the Altered Sites in vitro Mutagenesis System fromPromega (Madison, Wis.) as follows: A 2.3 kb Xba I fragment of Lyn Bfrom Lyn-pCDM8 was ligated into the Xba I site of pAlter-1. Mutagenesiswas carried out according to the manufacturer's protocol, using theampicillin repair primer provided in the kit and a Lyn single mutationantisense oligo: (5′-GCCAGGCTTGAGGGTCCTTACAGCCACTTTTGTGC-3′) (SEQ IDNO:8) to convert TTC (Lys) → TCC (Arg). The mutant Lyn B was digestedwith Xho I and BstB I and ligated back into pZeo.

Transfection of CHO Cells. Using Lipofectin reagent-mediatedtransfection (Life Technologies), pSVL constructs of the α, β and γsubunits of rat FcεRI, along with pSV2neo had been previously introducedinto CHO cells and a clone expressing a high number of receptors wasfrozen. After thawing, expression of receptors decreased rapidly withtime in culture so the culture was re-cloned by incubating the cellswith fluorescein-conjugated IgE and sorting on a fluorescence activatedcell sorter. The 1% of cells expressing the highest number of receptorswere re-sorted on 96-well plates at 0.5/cell per well. Fifty survivingclones were screened for expression of receptors by growing the cells toconfluence and sensitizing them with [¹²⁵I]-mouse IgE. The washed,adherent cells were solubilized with boiling SDS sample buffer and theIgE in the extract was quantitated by γ-counting. Of five highexpressing clones, one (CHO-B12) proved highly stable and was used forall subsequent studies. CHO-B12 cells were cryopreserved by freezing in5% dimethylsulfoxide/95% growth media (higher concentrations ofdimethylsulfoxide caused a rapid decline in FcεRI). The cells wereelectroporated (0.4 cm gap cuvettes, 200 V, 500 μF) in the presence ofone of several rat Lyn-pZeo constructs or empty pZeo vector which hadbeen linearized by digestion with Eco 57I. To select resistant clones,the media was supplemented with 250 μg/ml zeocin at 72 hr posttransfection.

Baculovirus expression of human Lyn B. The human Lyn B cDNA (1.5 Kb) wasexcised from pSVL by Xba I digestion and ligated into the homologous NheI site of pBlueBac. Sf9 cells were co-transfected with wild type AcMNPVDNA and the Lyn construct to generate recombinant Lyn baculoviruses.Adherent Sf9 cells were infected with plaque-purified baculovirus at amultiplicity of infection of 0.4. After 48 hrs, the cells were lysed in0.1% NP-40 buffer containing protease and phosphatase inhibitors.Western blotting with anti-PY indicated that the Lyn B protein wasphosphorylated on tyrosine as it was produced in the insect cells.

Stimulation of Cells. CHO cells to be stimulated with antigen weresensitized overnight with [¹²⁵I]-labeled mouse anti-DNP mouse IgE,washed thrice in buffer A (150 mM NaCl/5 mM KCl/25 mM Pipes, pH 7.2)plus 0.1% (w/v) gelatin and 5.4 mM dextrose and resuspended at 1×10⁷cells per ml. DNP6-BSA was added as a 5× stock solution to 5×10⁶ cellsat 37° C. for various time periods. CHO cells stimulated with IgEoligomers were incubated with various concentrations at 37° C. forvarious time periods.

Solubilization and Immunoprecipitation. After stimulation, the receptorswere solubilized in 0.05% Triton X-100 (3). For immunoprecipitation,anti-mouse or anti-rat IgE antibody was pre-bound to 30 μl proteinA-sepharose beads overnight in borate-buffered saline, pH 8, containing0.1% gelatin. The beads were recovered by centrifugation and combinedwith the lysates (“pre-cleared” with 100 μl protein A-sepharose beadsovernight) for two hours. After re-centrifnigation, theimmunoprecipitates were washed four times as previously described (3)and the bound proteins were released by boiling in SDS sample buffer forfive minutes.

Quantitation of Phosphorylation of Receptors. Immunoprecipitatedreceptors were separated by electrophoresis in SDS on 10% polyacrylamidegels equilibrated with Tricine and the phosphorylated proteins weredetected with an anti-PY antibody and an Enhanced Chemiluminescentdetection system (Amersham, Arlington Heights, Ill.) (25).Autoradiographs of Western blots were quantitated by computerizeddensitometry (Molecular Dynamics, Sunnyvale, Calif.). Three steps weretaken to ensure equal numbers of receptors were being compared in thosestudies in which cells co-transfected with inactive forms of Lyn werecompared to cells that had not been co-transfected. First, the cellswere incubated with IgE that had been labeled with [¹²⁵I] and equalnumbers of counts were loaded per lane. Second, one lane on each gel wasloaded with the same amount of phosphorylated human Lyn B to correct fordifferences in transfer, antibody staining, washing, etc. Third, theprimary anti-PY blots were stripped and reprobed with an antibody (JRK)to the β chain of the receptor (26) and densitometric analysis wasrepeated. The densitometric values from the primary anti-PY blots werethen corrected for any differences in anti-PY staining or loading ofreceptors. In separate experiments, the linearity of antibody staining(anti-PY, anti-β) was verified by loading increasing amounts of anappropriate protein extract and quantitating the band intensity.

Quantitation of Lyn. To quantitate the relative amounts of Lyn, wholecell lysates containing either 7×10⁴ or 1.6×10⁵ cell equivalents wereprepared with SDS for each transfectant. Depending on which moleculeshad been transfected, the samples were separated on 8% (Lyn B, RK Lyn),10% (CHO-B12, pZeo) or 4-20% (unique Lyn A) Tris-Glycine gels andblotted with an anti-Lyn antibody and an HRP-conjugated anti-rabbitsecondary antibody. One lane on each gel was loaded with a fixed amountof human Lyn B (above). The densitometric readings for the bandscorresponding to Lyn were normalized relative to the human Lyn Bstandard.

Quantitation of FcεRI. CHO cells were suspended at a concentration of510⁶ per ml and incubated with 5 μg/ml of [¹²⁵I]-labeled IgE for onehour at 37°. Nonspecific binding was evaluated by preincubating thecells with a 10-fold excess of unlabeled IgE for 30 minutes at 37°.Cells were separated from unbound IgE by pelleting through phthalate oil(15, 27).

Subcellular Fractionation. CHO cells were sonicated and the 140,000× gsupernatant (cytosolic fraction) and pellet (membrane fraction) wereprepared from the post-nuclear supernatant as previously described (28).Membrane proteins were solubilized in 0.5% Triton X-100 for 30 minutesat 4° C. Each subcellular fraction was treated with an equal volume ofboiling 2× SDS sample buffer for five minutes prior to gelelectrophoresis.

In vitro transcription/translation. Coupled in vitrotranscription-translation reactions were conducted with [³⁵S]-Cysaccording to the manufacturer's recommendation (T3 TnT® CoupledReticulocyte Lysate Systems, Promega). The reaction mixture containsMg²⁺, ATP and NaCl in a neutral pH buffer and can support a kinasereaction.

Yeast Two-hybrid Studies. Initial identification of potentiallyinteracting domains was conducted by co-transforming constructscontaining the cytoplasmic domains of the FcεRI fused to the bindingdomain of the Gal 4 transcription factor with constructs containing Lynor various mutated forms of Lyn fused to the activation domain of Gal 4(11) (FIG. 1).

The nucleotide sequence coding for the amino- and carboxy-terminalcytoplasmic domains of the β subunit of the rat IgE receptor, β_(N) andβ_(C), respectively, were subcloned into pAS2 to generate Gal 4 DNAbinding domain fusion proteins. Unfortunately, both fusion proteinsautonomously activated the reporter genes. This is presumably due to theacidic hemagglutinin epitope located between the Gal4 DNA binding domainand the inserted proteins (29). However, the fusion protein containingthe cytoplasmic domain of the γ subunit was not autonomously active.Therefore, nucleotide sequences coding for β_(N) and β_(C) weresubcloned into the newly developed vector, pAS-2-1, which is similar topAS2, but has the acidic hemagglutinin epitope removed. NeitherpAS2-1-β_(C) or pAS2-1-β_(N) were autonomously active.

The activities of the His and LacZ reporter genes in CG1945 yeasttransformants expressing Lyn and β_(N), β_(C) or γC were tested asdescribed herein. Both the full length and unique domain of both Lyn Aand Lyn B interacted directly with β_(C). However, the interaction wasmuch weaker than the interaction detected between the p53 and SV40fusion proteins used as a positive control. Thus, per μg DNA,co-transformation with p53 and SV40 resulted in more colonies onHis-deficient medium (SD-3) and rapid growth into large colonies. All ofthe colonies containing p53 and SV40 rapidly turned blue. In contrast,co-transformation with the Lyn and β_(C) constructs resulted in fewercolonies and slower growth on His-deficient medium and only the largecolonies turned blue. No interaction was detected between β_(N) or γ_(C)with any forms of Lyn in this assay.

To quantitate the interaction between Lyn and β_(C) or β_(N), theβ-galactosidase activity of these co-transformants in yeast strain Y187was measured in a liquid assay. In addition to the full-length Lyn andthe construct containing only the unique domain, a series of Lyn mutantswas tested on the basis of results of Pleiman, et al. (30) and TimsonGauen et al. (31). The negative control in this experiment was the 40amino acid residues (from 27 to 66; SEQ ID NO:5) of Lyn A fusedout-of-frame to the Gal 4-AD (pACT-27-66-OOF). These studies showed thatthe activity of the LacZ reporter gene from co-transformants with theunique domain of either Lyn A or B was as high as the activity fromco-transformants with the full-length form of either Lyn. These valuesare three-fold higher than those from the negative controlpACT-27-66-OOF. Consistent with the result from CG1945 strain, theinteraction between Lyn and β_(C) is weaker (on the basis of theβ-galactosidase activity, only 1 per cent as strong) than that betweenp53 and SV40. Co-transformants containing Lyn amino acid residues 1-10,1-27 or 27-66 produced slightly higher amounts of β-galactosidase thanthe negative control. No interaction between Lyn and β_(N) was detected.

Characterization of Transfected FcεRI in CHO Cells. A clone oftransfected CHO cells that stably expressed ˜170,000 receptors per cell(CHO-B12) (Table 2) was further characterized. When immunoblotted withanti-human Lyn antibody, extracts of these cells, like those of theuntransfected CHO cells, show a weakly reactive component at ˜58 kDa,i.e., slightly greater than the apparent molecular mass of 53 and 56 kDaobserved for rat Lyn. There was no reactivity with a panel of antibodiesto human c-Src, Fyn or c-Yes. Cells from the B12 clone were incubatedwith anti-DNP-specific mouse IgE and, after solubilization withdetergent, the bound (unaggregated) receptors were immunoprecipitatedwith goat anti-mouse IgE. Upon western blotting with anti-PY, noevidence for phosphorylation was observed. When the cells were incubatedwith multivalent antigen (DNP-BSA) prior to solubilization,phosphorylation of tyrosines on the β and γ subunits of the transfectedreceptors was observed. Disaggregation of the receptors in vivo byaddition of hapten (DNP-caproic acid) after the exposure to DNP-BSA ledto the complete reversal of the antigen-induced phosphorylation ofreceptor tyrosines within <1 minute.

RBL cells can be stimulated either by aggregating receptor-boundmonomeric IgE with antigen or by incubating the cells with preformeddimers of IgE. In contrast, incubation of CHO-B12 cells with dimeric IgEfailed to induce detectable phosphorylation of the receptors. Theseresults are consistent with a limiting amount of protein tyrosine kinasebeing associated with the receptors in these cells.

Correlation Between Total Lyn and Phosphorylation of FcεRI. A series ofstable transfectants of the CHO-B12 cells with rat Lyn were isolated.The relative ratios of full-length Lyn/receptor of six clones (A6through D8) are shown in the upper part of column 5 of Table I.Subcellular fractionation of the transfected cells indicated that thetransfected full-length Lyn was expressed as a membrane-associatedprotein, as expected for a Src-family kinase (32).

The various transfectants were stimulated either with IgE dimers or withmonomeric IgE, followed by antigen, to examine the relationship betweenthe total cellular content of Lyn and the responsiveness of the cells.Care was taken to ensure equal numbers of receptors were being compared.

A good correlation between the amount of Lyn expressed and the amount ofreceptor tyrosine phosphorylation was seen with both the β and γsubunits upon aggregation of the receptors with antigen. Furthermore,all of the cells expressing transfected Lyn now responded to dimers ofIgE. More extensive phosphorylation was observed in those cells whosereceptors were aggregated with antigen rather than with dimers. However,the stimulation by dimers was more sensitive to the amount of Lynexpressed.

One clone, A11, in which the relative Lyn/receptor ratio wasexceptionally high, showed a significant degree of phosphorylation ofthe receptors even without stimulation. Western blotting of A11 lysatesrevealed a phosphorylated component with an apparent molecular mass of53 kDa (presumably Lyn), but no change in overall phosphorylation oftyrosines on other cellular proteins when compared to CHO-B12 lysates.

To control for differences in tyrosine phosphorylation that may havearisen due to zeocin resistance alone, CHO-B12 cells were transfectedwith pZeo vector and resistant colonies were isolated and expanded. Uponstimulation with 0.5 μg/ml trimeric IgE from 5 to 30 minutes, the sixzeocin resistant clones tested showed no significant differences inphosphorylation of the β and γ subunits of the receptor as compared toCHO-B12 cells. In a similar experiment, the responses to varying dosesof antigen (25-300 ng/ml) of three zeocin-resistant clones were comparedto CHO-B12 cells. A similar dose-dependence of phosphorylation of thereceptors was observed. No differences were noted in either themagnitude or pattern of total cellular proteins that became tyrosinephosphorylated. By Western blotting, the level of endogenous Lyn wasalso unchanged. Since the number of FcεRI on the pZeo transfectantsvaried between 80,000 and 150,000 (clones Z1 to Z6, Table 2), the degreeof phosphorylation was found to be independent of the number ofreceptors under the conditions used in this study.

Mapping The Site of Lyn-FcεRI Interaction by Competition. The presencein the CHO-B12 cells of an endogenous kinase (presumably Lyn) capable ofphosphorylating the receptor allowed for probing the site on Lyn whichinteracts with the FcεRI by a competition protocol. Cells weretransfected with domains of Lyn that would potentially interact with thereceptor but that were themselves catalytically inactive. Theresponsiveness of such transfectants to aggregation of their FcεRI wascompared either to CHO-B12 or to cells co-transfected with the “empty”pZeo vector.

Catalytically Inactive Lyn Kinase. A full length, catalytically inactiveLyn B kinase was prepared by mutating Lys279 to Arg (RK Lyn). In acoupled in vitro transcription-translation reaction, the wild-type Lynwas autophosphorylated, whereas the mutant Lyn was not.

Three stable transfectants expressing substantial amounts of the mutantLyn were isolated and assessed (clones RK 17, 21 and 26 in Table 2). Thecatalytically inactive Lyn was expressed largely or exclusively as amembrane anchored protein. On a per receptor basis, such stable RKLyn-FcεRI transfectants showed 20-75% less antigen-inducedphosphorylation of receptor tyrosines than cells transfected with thevector alone. Therefore, a single point mutation converted a constructthat stimulated phosphorylation of tyrosines on FcεRI to one thatinhibited it.

Unique Domain of Lyn Kinase. On the basis of the results from theyeast-two hybrid studies, the unique domain of Lyn A kinase wastransfected into receptor-containing cells (clones C6, U7 and U8 inTable 2). The isolated unique domain was expressed largely orexclusively in a membrane-anchored form. Upon stimulation withmultivalent antigen, a partial inhibition of phosphorylation of receptortyrosines was observed. A comparison of two clones expressing increasinglevels of the unique domain protein showed that increasing amounts ofthe competing domain led to increasing inhibition. With a weakerstimulus (IgE trimers), complete inhibition of phosphorylation of the βand γ chains was observed at early time points and at low concentrationsof stimulant.

Identifying portions of the unique domain of Lyn A and Lyn B with FcεRIsignal transduction inhibiting activity. On the basis of the knownsequence of the unique domain of Lyn A (amino acids 1-66) and Lyn B(amino acids 1-45), peptides having at least five contiguous amino acidsfrom these domain sequences can be produced according to peptidesynthesis protocols well known in the art and tested for the ability tobind the FcεRI βc according to protocols known in the art and asdescribed herein (e.g, the yeast two hybrid assay). The peptides whichbind the FcεRI βc can then be evaluated for the ability to inhibitsignal transduction through the receptor according to well known methodsand as described herein (e.g., inhibition of receptor phosphorylation ina transfection assay). For example, the peptides having amino acids 1-10(SEQ ID NO:1), 1-27 (SEQ ID NO:2) and 27-66 (SEQ ID NO:3), which areknown to bind FcεRI βc can be evaluated for the ability to inhibit thesignal transducing activity of the receptor by the methods herein. Inaddition, one or more amino acids can be deleted from these peptides orone or more amino acids can be added to these peptides and evaluatedaccording to the teachings herein for the ability to bind FcεRI βc andinhibit its signal transducing activity.

Treatment of an allergic disorder in a human with the nucleic acid orfusion protein of the present invention. Either 1) the nucleic acid ofthis invention, as naked DNA or carried in a vector in apharmaceutically acceptable carrier, or 2) the fusion protein of thisinvention, in a pharmaceutically acceptable carrier, can be administeredintranasally or by inhalation to a human subject diagnosed with anallergic disorder, in an amount and for a time interval determined byone of skill in the art to be effective in treating the allergicdisorder according to standard protocols for determining optimal dosagesand treatment regimens as described herein. Clinical parameters can bemonitored over time as described herein and the treatment can becontinued until the allergic disorder is improved or subsides. Treatmentcan be resumed upon the recurrence of symptoms or other clinicalindicators that an allergic disorder is being manifested.

Although the present process has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

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TABLE 1 CONSERVATIVE SUBSTITUTIONS Amino acids Shared properties whichare interchangeable for each other Neutral and hydrophobic Alanine (AlaA); Valine (Val V); Leucine (Leu L); Isoleucine (Ile I); Proline (ProP); Tryptophan (Trp W); Phenylalanine (Phe F); Methionine (Met M)Neutral and polar Glycine (Gly G); Serine (Ser S); Threonine (Thr T);Tyrosine (Tyr Y); Cysteine (Cys C); Glutamine (Gln Q); Asparagine (AsnN) Basic Lysine (Lys K); Arginine (Arg R); Histidine (His H) AcidicAspartic Acid (Asp D); Glutamic Acid (Glu E)

TABLE 2 CHO TRANSFECTANTS Lyn (Inact.) FcεRI × Lyn per per Name CloneInsert 10⁻⁵ FCεRI^(c) Lyn (End.) CHO—B12^(a) B12 NA^(b) 1.7 2.5 NALyn/B12 A6 Lyn B 1.0 1.0 NA Lyn/B12 A9 Lyn B 0.7 9.3 NA Lyn/B12 A11 LynB 1.3 66 NA Lyn/B12 D1 Lyn B 1.3 34 NA Lyn/B12 D7 Lyn B 1.3 3.2 NALyn/B12 D8 Lyn B 1.3 0.94 NA RK Lyn/B12 RK17 Inact.Lyn B 1.9 7.4 6.0^(d)RK Lyn/B12 RK21 Inact.Lyn B 1.8 4.6 4.2 RK Lyn/B12 RK26 Inact.Lyn B 1.424 12 Lyn C6 Unique Lyn A 1.2 5.9 5.0 unique/B12 Lyn U7 Unique Lyn A 1.00.83 0.42 unique/B12 Lyn U8 Unique Lyn A 1.7 1.9 0.76 unique/B12pZeo/B12 Z1 None 0.8 5.2 NA pZeo/B12 Z2 None 0.8 6.6 NA pZeo/B12 Z3 None1.0 5.0 NA pZeo/B12 Z4 None 0.8 6.8 NA pZeo/B12 Z5 None 1.5 2.8 NApZeo/B12 Z6 None 1.0 6.6 NA ^(a)A stable CHO FcεRI transfectant(CHO—B12) was generated by electroporation of FcεRI subunits (α, β, γ).Stable double transfectants, likewise generated electroporation, wereprepared by transfecting various Lyn constructs into CHO—B12 cells andselection with zeocin. Control cells, doubly transfected with FcεRI andempty pZeo vectors, were prepared by the same protocol. ^(b)NA, notapplicable ^(c)The values shown in this column are strictly relative andwere determined as follows. For each transfectant, the normalizeddensitometric readings of the total Lyn in a fixed number of cellequivalents was divided by the number of FcεRI per cell × 10⁻³. Forexample, for the first item in this column the corrected densitometricreading was 430.6. The latter divided by 170 # (FcεRI per cell × 10⁻³)equals 2.5. The quantitation of Lyn was done two to eight times for eachtransfectant; the enumeration of the FcεRI was done in duplicate.^(d)The amount of endogenous hamster Lyn (Lyn (End.)) and transfectedinactive Lyn (Lyn(Inact.)) expressed per cell was determined by Westernblotting of SDS lysates with anti-(human) Lyn. The values shownrepresent the average of two to eight separate determinations.

8 1 66 PRT Artificial Sequence Description of Artificial Sequence/Note =synthetic construct 1 Met Gly Cys Ile Lys Ser Lys Gly Lys Asp Ser LeuSer Asp Asp Gly 1 5 10 15 Val Asp Leu Lys Thr Gln Pro Val Arg Asn ThrGlu Arg Thr Ile Tyr 20 25 30 Val Arg Asp Pro Thr Ser Asn Lys Gln Gln ArgPro Val Pro Glu Ser 35 40 45 Gln Leu Leu Pro Gly Gln Arg Phe Gln Thr LysAsp Pro Glu Glu Gln 50 55 60 Gly Asp 65 2 45 PRT Artificial SequenceDescription of Artificial Sequence/Note = synthetic construct 2 Met GlyCys Ile Lys Ser Lys Gly Lys Asp Ser Leu Ser Asp Asp Gly 1 5 10 15 ValAsp Leu Lys Thr Gln Pro Val Pro Glu Ser Gln Leu Leu Pro Gly 20 25 30 GlnArg Phe Gln Thr Lys Asp Pro Glu Glu Gln Gly Asp 35 40 45 3 10 PRTArtificial Sequence Description of Artificial Sequence/Note = syntheticconstruct 3 Met Gly Cys Ile Lys Ser Lys Gly Lys Asp 1 5 10 4 27 PRTArtificial Sequence Description of Artificial Sequence/Note = syntheticconstruct 4 Met Gly Cys Ile Lys Ser Lys Gly Lys Asp Ser Leu Ser Asp AspGly 1 5 10 15 Val Asp Leu Lys Thr Gln Pro Val Arg Asn Thr 20 25 5 40 PRTArtificial Sequence Description of Artificial Sequence/Note = syntheticconstruct 5 Thr Glu Arg Thr Ile Tyr Val Arg Asp Pro Thr Ser Asn Lys GlnGln 1 5 10 15 Arg Pro Val Pro Glu Ser Gln Leu Leu Pro Gly Gln Arg PheGln Thr 20 25 30 Lys Asp Pro Glu Glu Gln Gly Asp 35 40 6 39 DNAArtificial Sequence Description of Artificial Sequence/Note = syntheticconstruct 6 cgggcggctc gatgggatgt attaaatcaa aaaggaaag 39 7 36 DNAArtificial Sequence Description of Artificial Sequence/Note = syntheticconstruct 7 cggcggctcg agctagtccc cttgctcctc tggatc 36 8 35 DNAArtificial Sequence Description of Artificial Sequence/Note = syntheticconstruct 8 gccaggcttg agggtcctta cagccacttt tgtgc 35

What is claimed is:
 1. An isolated nucleic acid selected from the groupconsisting of: a) a nucleic acid encoding the amino acid sequenceconsisting of amino acids 1-66 of the human tyrosine kinase, Lyn A (SEQID NO:1); b) a nucleic acid encoding the amino acid sequence consistingof amino acids 1-10 of the human tyrosine kinase, Lyn A (SEQ ID NO:3);and c) a nucleic acid encoding the amino acid sequence consisting ofamino acids 27-66 of the human tyrosine kinase, Lyn A (SEQ ID NO:5). 2.A vector comprising the nucleic acid of claim
 1. 3. The nucleic acid ofclaim 1 in a pharmaceutically acceptable carrier.
 4. A cell comprisingthe vector of claim
 2. 5. An isolated nucleic acid encoding the aminoacid sequence of amino acids 1-45 of the human tyrosine kinase, Lyn B(SEQ ID NO:2).
 6. A vector comprising the nucleic acid of claim
 5. 7.The nucleic acid of claim 5 in a pharmaceutically acceptable carrier. 8.A cell comprising the vector of claim
 6. 9. An isolated nucleic acidencoding the amino acid sequence of a fusion protein, comprising i) anucleic acid encoding a ligand which binds to and is internalized bycells which express a high affinity receptor for IgE on the surface andii) a nucleic acid selected from the group consisting of: a) a nucleicacid encoding the amino acid sequence of amino acids 1-66 of the humantyrosine kinase, Lyn A (SEQ ID NO:1); b) a nucleic acid encoding theamino acid sequence of amino acids 1-10 of the human tyrosine kinase,Lyn A (SEQ ID NO:3); c) a nucleic acid encoding the amino acid sequenceof amino acids 1-27 of the human tyrosine kinase, Lyn A (SEQ ID NO:4);d) a nucleic acid encoding the amino acid sequence of amino acids 27-66of the human tyrosine kinase, Lyn A (SEQ ID NO:5); and e) a nucleic acidencoding a peptide comprising any five or more contiguous amino acids ofthe amino acid sequence of amino acids 1-66 of the human tyrosinekinase, Lyn A, wherein the peptide has the same IgE (FcεRI) receptorbinding activity as the amino acid sequence encoded by the nucleic acidsequence as set forth in (a), (b), (c) or (d).
 10. A vector comprisingthe nucleic acid of claim
 9. 11. The nucleic acid of claim 9 in apharmaceutical carrier.
 12. The nucleic acid of claim 9, wherein theligand is selected from the group consisting of IgE and c-Kit.
 13. Acell comprising the vector of claim
 10. 14. An isolated nucleic acidencoding the amino acid sequence of a fusion protein, comprising i) anucleic acid encoding a ligand which binds to and is internalized bycells which express a high affinity receptor for IgE on the surface andii) a nucleic acid selected from the group consisting of: a) a nucleicacid encoding the amino acid sequence of amino acids 1-45 of the humantyrosine kinase, Lyn B (SEQ ID NO:2); and b) a nucleic acid encoding apeptide comprising any five or more contiguous amino acids of aminoacids 1-45 of the human tyrosine kinase, Lyn B (SEQ ID NO:2), whereinthe peptide has the same IgE (FcεRI) receptor binding activity as theamino acid sequence encoded by the nucleic acid sequence as set forth in(a).
 15. A vector comprising the nucleic acid of claim
 14. 16. Thenucleic acid of claim 14 in a pharmaceutically acceptable carrier. 17.The nucleic acid of claim 14, wherein the ligand is selected from thegroup consisting of IgE and c-Kit.
 18. A cell comprising the vector ofclaim 15.